Recombinant poxviruses are widely used to express foreign antigens in infected cells. Moreover, recombinant poxviruses are very promising vaccines for inducing an immune response against the foreign antigen expressed from the poxvirus vector. Most popular are avipoxviruses on the one hand and, on the other hand, vaccinia viruses, in particular modified vaccinia virus Ankara (“MVA”). MVA is related to vaccinia virus, a member of the genus Orthopoxvirus in the family of Poxyiridae.
MVA has been generated by 516 serial passages on chicken embryo fibroblasts of the Ankara strain of vaccinia virus (CVA) (for review, see Mayr et al. (1975) Infection 3, 6-14). As a consequence of these long-term passages, the resulting MVA virus deleted about 31 kilobases of its genomic sequence and, therefore, was described as highly host cell-restricted to avian cells (Meyer et al. (1991) J. Gen Virol. 72, 1031-1038). It was shown, in a variety of animal models, that the resulting MVA was significantly avirulent (Mayr and Danner (1978) Dev. Biol. Stand. 41, 225-34). Additionally, this MVA strain has been tested in clinical trials as a vaccine to immunize against the human smallpox disease.
For the expression of heterologous genes in pox viruses, only a few promoters are known, such as the 30K and 40K promoters (see for example U.S. Pat. No. 5,747,324), a strong synthetic early/late promoter (see for example Sutter et al. (1994) Vaccine 12, 1032-1040), the Pr7.5 promoter (7.5K promoter; see for example Endo et al. (1991) J. Gen. Virol 72, 699-703) and the promoter derived from the cowpox virus A-type inclusion (ATI) gene (PrATI, Li et al. (1998) J. Gen. Virol. 79, 613). All of these promoters have been used in recombinant vaccinia viruses to express heterologous genes and were shown to express said genes resulting in the production of the protein encoded by the heterologous gene. However, there is still a general need for alternative promoters, specifically strong promoters, in vaccinia viruses.
MVA is thus widely used as a vaccine as well as an expression system to make large amounts of recombinant protein. Especially in its use as vaccine, it is desirable to produce high levels of antigen, as the desired immune response elicited depends in part on the amount of protein produced following administration of the MVA-containing vaccine. Up to now, enhancing the desired immunogenicity of a protein has been accomplished in a number of ways. First, when administering the naked antigenic protein as a vaccine, larger amounts of the relevant protein may be introduced into the subject to be vaccinated. In a vaccine based on a recombinant expression system, i.e. a vaccine in which the encoding nucleic acid is administered, for example, in a poxvirus vector such as MVA, and the actual immune response is elicited by a protein expressed from this nucleic acid, it is also possible to enhance immunogenicity by enhancing the transcription of the antigen (Wyatt et al. (2008) Vaccine 26, 486-493). It is also possible to enhance the translation of already transcribed RNA. Finally, immunogenicity can be enhanced by enhancing the presentation of the antigen to the immune system.
One problem frequently encountered in designing viral recombinant expression systems, especially for use as vaccines, is that the constructs often lack stability at the nucleic acid level. Further, the nucleic acid is frequently subject to undesired recombination within the viral vector, which often reduces, and in some cases even abrogates, the capacity of the viral vector to produce the desired immunogenic peptide in the vaccinated host.
It is therefore an aim to overcome these problems while retaining the high degree of protein expression required for eliciting a sufficient immunogenic response.